Experimental Cell Research 252, 449 – 463 (1999) Article ID excr.1999.4650, available online at http://www.idealibrary.com on
Phosphatases Involved in Modulation of Gap Junctional Intercellular Communication and Dephosphorylation of Connexin43 in Hamster Fibroblasts: 2B or Not 2B? 1 Ve´ronique Cruciani,* Olav Kaalhus,† and Svein-Ole Mikalsen* ,2 *Department of Environmental and Occupational Cancer and †Department of Biophysics, Institute for Cancer Research, The Norwegian Radium Hospital, N-0310 Oslo, Norway
INTRODUCTION 12-O-Tetradecanoylphorbol-13-acetate (TPA) caused strong suppression of gap junctional intercellular communication, altered phosphorylation status of the gap junction protein, connexin43, and disappearance of immunorecognizible connexin43-containing gap junction plaques in V79 fibroblasts. When TPA was removed, all parameters normalized during a 3- to 4-h period. The normalizations were independent of protein synthesis, suggesting the possible involvement of phosphatases. None of the phosphatase inhibitors okadaic acid, calyculin A, cyclosporin A, or FK506 affected intercellular communication or connexin43 phosphorylation status on their own. In sequential exposures to TPA and phosphatase inhibitors, only the proteinphosphatase 2B (PP2B) inhibitors cyclosporin A and FK506 delayed the recovery of the studied parameters. Rapamycin binds to the same set of proteins as does FK506, but without inhibiting PP2B. Rapamycin did not affect the recovery of intercellular communication, but it delayed the normalization of connexin43 band pattern and immunorecognition of gap junction plaques. Dephosphorylation of immunoprecipitated connexin43 was studied using PP1, 2A, 2B, and 2C. PP2A was the most efficient (by 100-fold on a molar basis). Connexin43 immunoprecipitated from TPA-exposed cells was a poor substrate for PP1, 2B, and 2C. Thus, PP2B appeared to play a role in normalization of intercellular communication, but not necessarily in direct dephosphorylation of connexin43. Peptidylprolyl isomerase activity of cyclosporin/FK506/rapamycin-binding proteins may promote the dephosphorylation of connexin43 in cells. © 1999 Academic Press Key Words: gap junctional intercellular communication; connexin43; phorbol ester; protein phosphatases; phosphatase inhibitors; immunophilins.
1 Our apologies to Hamlet, Prince of Denmark, and William Shakespeare. 2 To whom correspondence and reprint requests should be addressed. Fax: 1 47 2293 5767. E-mail:
[email protected].
Gap junctional intercellular communication (GJIC) is of importance in development, differentiation, and growth control [1– 4]. It is also important in the control of other physiological processes, e.g., synchronization of the heartbeat, and production and secretion of insulin [5]. Mutated gap junction proteins or aberrant GJIC are found in an increasing number of pathological conditions, including cancer [1, 2, 4, 6]. It is therefore of importance to understand the regulation of GJIC. The gap junction channels are assembled from a family of proteins called connexins. Except for the smallest connexin, connexin26 (Cx26), they are phosphoproteins or contain potential phosphorylation sites. Connexin43 (Cx43) is one of the major connexins in the body and is found in many organs and cell types [1]. Cultured cells have a tendency to express Cx43, often concurrently with a shutdown in the expression of other connexins [1]. Many growth factors and hormones influence GJIC [7]. Most signal transduction pathways utilize protein kinases at one or more steps. Several kinases have been implicated in phosphorylation of connexins. The most studied connexin in this respect is Cx43. Activation of protein kinase C (PKC) by 12-O-tetradecanoylphorbol-13-acetate (TPA) and mitogen-activated protein kinase by epidermal growth factor (EGF) are found to induce changes in the Cx43 band pattern and cause decreased GJIC in some cell systems [8 –11]. Since kinases have been intimately implicated in the regulation of GJIC and phosphorylation of Cx43, it should be expected that also phosphatases must be acting in these processes. In addition, Crow et al. [12] found a rapid phosphorylation/dephosphorylation cycle after the synthesis of Cx43. However, the involvement of phosphatases in the regulation of GJIC and phosphorylation status of Cx43 has been relatively little studied. Calyculin A and okadaic acid are potent inhibitors of protein phosphatase types 1 and 2A (PP1 and PP2A),
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two of the major classes of phosphoserine- and phosphothreonine-specific phosphatases in cells. Okadaic acid does not affect GJIC in early passage Syrian hamster embryo (SHE) cells (mainly consisting of fibroblasts), either alone or after exposure to TPA [13]. Okadaic acid, calyculin A, vanadate, molybdate, and vanadyl sulfate minimally affect GJIC and Cx43 band pattern in SHE cells [8]. Also in other cell types, the Cx43 band pattern is not changed by okadaic acid or calyculin A [9, 14 –16], although the incorporation of phosphate in Cx43 might increase [9]. This may indicate that the phosphorylation status of Cx43 is relatively stable under nonstimulating conditions or that the phosphorylation sites are not good substrates or not available for these phosphatases. Even after stimulation with TPA, the rate of normalization of GJIC in SHE cells was not affected by okadaic acid and calyculin A [8]. In contrast, Lau et al. [9] found okadaic acid to decrease the rate of normalization after GJIC had been suppressed by EGF in rat liver epithelial T51B cells. One interesting difference between early passage SHE cells [8, 11] and the T51B cell line [9] is that SHE cells show only very small changes in phosphorylation status of Cx43 after exposure to TPA, while T51B cells have prominent changes in Cx43 phosphorylation status following treatment with EGF. Thus, the contrasting results of okadaic acid in stimulated cells could be due to the differential response of Cx43 in the two systems. Furthermore, the possible involvement of PP2B or PP2C in regulation of GJIC or dephosphorylation of Cx43 has not been investigated. We have therefore compared the response of V79 cells with that of SHE cells to inhibitors of PP1, PP2A, and PP2B. PP2B is inhibited by the immunosuppressants cyclosporin A and FK506 due to a compound-induced complexing of cyclophilins or FK506-binding proteins (FKBPs) to PP2B [17]. Cyclophilins and FKBPs (collectively known as immunophilins) possess peptidyl-prolyl cistrans isomerase (PPIase; also called rotamase) activity that is inhibited by the compounds [18]. Rapamycin is another immunosuppressant that competes with FK506 for binding to FKBPs. Rapamycin inhibits the PPIase activity, but it does not inhibit PP2B [17, 19]. We also investigated the efficiencies of PP1, PP2A, PP2B, and PP2C in dephosphorylation of Cx43 immunoprecipitated from control cells and cells treated with TPA. MATERIAL AND METHODS Chemicals. Calyculin A, okadaic acid, cyclosporin A, cypermethrin, and protein phosphatase inhibitor-2 were from Calbiochem (La Jolla, CA). FK506 was obtained from Alexis Corporation (La¨ufelfingen, Switzerland). Rapamycin, TPA, Lucifer yellow CH, leupeptin, pepstatin A, and human recombinant FKBP were from Sigma (St. Louis, MO). Pefabloc, aprotinin, calmodulin, alkaline
phosphatase, PP1g (catalytic subunit, recombinant human form), and PP2A (catalytic subunit purified from bovine heart) were from Boehringer (Mannheim, Germany). PP2B (holoenzyme purified from bovine brain), PP2Ca (recombinant human form), anti-PP1a, and anti-phosphotyrosine (4G10) were from Upstate Biotechnology (Lake Placid, NY). Anti-PP2A and anti-PP2Ba were from Calbiochem, and anti-PP1a (C-19), anti-PP1b (C-20), anti-PP1g (C-19), and anti-PP2A (G-20) were bought from Santa Cruz Biotechnology (Santa Cruz, CA). Cell cultures. The immortal Chinese hamster lung fibroblast cell line, V79, was grown in Dulbecco’s modified Eagle’s medium (DMEM; BioWhittaker, Walkersville, NY) supplemented with 7.5% fetal calf serum (Life Technologies, Paisley, Scotland) at 37°C in a humidified 10% CO 2 atmosphere. Early passage SHE cells made from Syrian hamster embryos at 14 days of gestation [8] were grown in DMEM with 10% fetal calf serum. SHE cells can be grown for 10 –15 passages before senescence. No antibiotics were used for any of the cell cultures. In experiments where conditioned medium was used, it was produced by parallel cell cultures. Gap junctional intercellular communication. GJIC was measured by dye transfer after microinjection of single cells [8]. Sequential exposures to TPA and phosphatase inhibitors (or other compounds) were used in most experiments. The cells were exposed to TPA for 30 min in fresh growth medium (i.e., DMEM with serum) or conditioned growth medium and rinsed twice in the appropriate medium, and the second compound was added in the appropriate medium, before GJIC was assayed at the given times. Control cultures were similarly treated with growth medium without compounds, but including vehicle (dimethyl sulfoxide or ethanol). It was never observed that the vehicle compounds affected the results compared with control without vehicle. The results are shown as means 6 SEM for n injections and are expressed in percentage relative to the corresponding time-matched growth medium-treated control. The statistical significances were calculated by a computerized ANOVA with Student–Newman–Keuls posttest on timematched data points using the real number of dye-coupled cells. Western blotting. Cells were exposed as described above. Western blotting was done after electrophoretic separation of proteins in 8% polyacrylamide gels [8]. The anti-Cx43 antiserum [8, 11, 20 –23] is directed against the 19 C-terminal amino acids of rat Cx43. The peptide was added a tyrosine N-terminally for coupling purposes. Immunofluorescence. Immunofluorescence experiments were done as described by Cruciani et al. [11], except that the cells were fixed in formaldehyde (3% in phosphate-buffered saline). For the micrographs, the only selection criterion was that the cells had a reasonable confluence; otherwise the micrographs were taken at random places in the dishes. The experiments were performed twice, and in each experiment around 10 –12 micrographs were taken per data point. The micrographs were copied at a final magnification of 13003. The number of gap junction plaques was counted in wellfocused cell– cell borders and the lengths of these cell– cell borders were measured. The number of gap junction plaques per unit length of membrane was calculated and expressed as percentage of concurrent time-matched controls (i.e., exposed to growth medium only). Statistical significances for the gap junction density (number of gap junctions per unit length of membrane) were calculated on basis of mean 6 SD for n micrographs using ANOVA with Student–Newman–Keuls posttest on time-matched data points. For analysis of the size distribution of gap junctions, the gap junctions were divided in three size classes (,1, 1–2, and .2 mm on the micrographs at 13003 magnification). The distribution index I was defined as the weighted mean of the size, letting the size classes carry the weight 1, 2, and 3, respectively. The distribution variance V was defined as the variance of I. Systematic differences between the experiments were eliminated by using the experiment number as a separate independent variable. Statistical significances were cal-
PHOSPHATASES AND CONNEXIN43 culated by ANOVA and Student–Newman–Keuls posttest, and P , 0.05 was regarded as significant. Measurement of phosphatase activities. PP1 and PP2A activities were measured by a [ 32P]phosphorylase a kit (Life Technologies) as described by the supplier. [g- 32P]ATP was obtained from Amersham. The assay buffer was added 0.1% Triton X-100. The [ 32P]phosphorylase a phosphatase activity that could be inhibited by inhibitor-2 was defined as PP1 activity. PP2B was determined by a microplate spectrophotometrical phospho-RII phosphatase kit (Biomol, Plymouth Meeting, PA). RII is a peptide from the regulatory subunit of protein kinase A. The activity that could be inhibited by the combination of FK506 and FKBP was regarded as PP2B. The phosphatase activity inhibited by 10 –100 nM calyculin A or okadaic acid was defined as the sum of PP1 and PP2A. PP2C activity was determined as Mg 21-dependent dephosphorylation of 32P-labeled casein as described by McGowan and Cohen [24]. Casein (Sigma) was labeled by the use of the catalytic subunit of protein kinase A (Sigma). Immunoprecipitation of Cx43 and phosphatase treatment. Immunoprecipitation was done as described by Mikalsen et al. [20]. Cells grown in 50-mm dishes were scraped into 200 ml of immunoprecipitation buffer and sonicated. An aliquot (10 ml) was taken for a direct Western blot before dilution of the homogenates followed by immunoprecipitation. The last washing of the pellet was performed in the buffer of the corresponding phosphatase (as advised by the suppliers for PP1, PP2A, and PP2C and as described by Su et al. [19] for PP2B but without addition of okadaic acid). Phosphatases were then added to the samples (for PP2B also 25 mg/ml of calmodulin was added) and incubated at 37°C for 3 h. At the end of this treatment, the supernatant was removed and 20 –30 ml of electrophoresis sample buffer was added per pellet. Western blotting was performed after heating the samples at 95°C for 5 min. The phosphatases were checked for enzyme activity as suggested by the suppliers. In the present system, actin migrates immediately above the Cx43 area. It was therefore verified that a broad specificity polyclonal anti-actin rabbit antiserum (Sigma A5060) did not recognize actin in Cx43 immunoprecipitates and that anti-Cx43 antiserum did not recognize actin from rabbit muscle (Sigma A2522). Thus, it is unlikely that actin interferes with the present conclusions.
RESULTS
GJIC Consistent with previous results [11, 25, 26], TPA caused strong inhibition of GJIC in V79 cells. After 30 min of exposure to 5 nM TPA, GJIC was suppressed to about 15% of normal level. When TPA was removed, GJIC was maintained at the same low level for about 2 h before a normalization started, and control level was reached at about 4 h after removal of TPA (Fig. 1) In the continued presence of TPA (5 nM), GJIC was at approximately 50% of control after 8 h of exposure (not shown). Under the present conditions, the procedure of washing cells with fresh growth medium or conditioned medium did not cause any change in GJIC (not shown), while GJIC is changed in V79 cells added fresh growth medium after 24 h serum starvation (V. Cruciani and S.-O. Mikalsen, unpublished data). The normalization of GJIC after removal of TPA was not delayed by cycloheximide in the period between 0 and 3.5 h after removal (Fig. 1), consistent with the results of Yamasaki et al. [27]. During 4- to 6-h expo-
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FIG. 1. Cycloheximide did not affect normalization of GJIC in V79 cells after preexposure to TPA. Cells were (h, dashed curve) preexposed to 5 nM TPA for 30 min, rinsed, and added growth medium. Alternatively, cells were (‚) pretreated with growth medium for 30 min, rinsed, and exposed to 10 mM cycloheximide or (Œ) preexposed to 5 nM TPA for 30 min, rinsed, and exposed to 10 mM cycloheximide. In all cases, GJIC was assayed at the indicated times after rinsing (n 5 22–73). In these experiments, the control level of GJIC as expressed in number of dye-containing cells per injection increased from 19.8 6 1.3 (mean 6 SEM; n 5 39) at 0 h to 26.5 6 2.2 (n 5 30) at 6 h. For each point of time, the control level was defined as the 100% level of GJIC. The data are presented as described under Materials and Methods. A statistical significance between the recovery curves of TPA preexposed cells with and without cycloheximide only occurred at the 5- and 6-h points (P , 0.05), but cycloheximide alone is also significantly different from medium-treated controls at 4, 5, and 6 h (P , 0.05).
sures, cycloheximide alone caused a lowering of GJIC (Fig. 1). This was probably related to the inhibition of protein synthesis that interfered with the turnover of Cx43. We conclude that protein synthesis is not needed for the normalization of GJIC after exposure to TPA. We have studied here if protein phosphatases are involved in the normalization. The dose responses on GJIC in V79 cells were studied after 30 min exposures to okadaic acid, calyculin A, cyclosporin A, and FK506. The two former are inhibitors of PP1 and PP2A, and the two latter are inhibitors of PP2B. Okadaic acid was used up to 100 nM. Higher concentrations were cytotoxic, causing the cells to detach. Calyculin A was very toxic and could not be used above 3 nM. Cyclosporin A and FK506 were used up to 300 nM without showing any toxicity. None of the compounds inhibited the dye transfer in V79 cells after 30-min exposures (not shown). The time responses of the highest concentrations of the compounds were then studied during exposures up to 4 h. None of the compounds caused a significant change in GJIC (Fig. 2A). Subsequently, V79 cells were preexposed to 5 nM TPA for 30 min before rinsing in growth medium, followed by exposure to phosphatase inhibitors for periods up to 6 h. The PP1 and PP2A inhibitors, okadaic acid and calyculin A, did not affect the normalization of GJIC after removal of TPA (Fig. 2B). Okadaic acid was also added together with TPA during a 30-min period before rinsing, and the cells recovered in the presence
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FIG. 2. Effects on GJIC of phosphatase inhibitors. (A) Time response on GJIC in V79 cells by ({) 3 nM calyculin A, (‚) 30 nM okadaic acid, (E) 300 nM cyclosporin A, and (■) 300 nM FK506, shown as percentage of control (mean 6 SEM, n 5 29 –90 injections per point). The cells were treated for 30 min with fresh growth medium before rinsing and addition of the compounds. GJIC was then assayed at the indicated times. (B) Influence of phosphatase inhibitors during the recovery period after preexposure of V79 cells to TPA. Cells were preexposed to 5 nM TPA for 30 min, rinsed, and added (h, dashed curve) growth medium only or ({) 1 nM calyculin A, (‚) 100 nM okadaic acid, (E) 300 nM cyclosporin A, or (■) 300 nM FK506 for the indicated periods before measuring GJIC (n 5 29 – 83). Compared to unexposed controls, P , 0.001 was generally obtained at approximately 70% of control value. When the sequential exposures of TPA and phosphatase inhibitors were compared to the recovery in cultures sequentially exposed to TPA and growth medium only, P , 0.001 was obtained at 3 and 3.5 h of exposure to FK506 and 3 h of exposure to cyclosporin A; and P , 0.01 was obtained at 4 h of exposure to FK506 and 3.5 and 4 h of exposure to cyclosporin A.
of okadaic acid alone. Again no difference was found between the cultures with and without okadaic acid (not shown). In contrast, both cyclosporin A and FK506 delayed the normalization by about 1 h (Fig. 2B). It was investigated if cyclosporin A and FK506 could inhibit the TPA-induced decrease in GJIC in V79 cells. The cells were exposed for 1 h to these compounds (300 nM), with 5 nM TPA present during the last 30 min. The compounds did not affect the TPA-induced decrease in GJIC (not shown). In contrast to V79 cells, SHE cells show minimal changes in Cx43 band pattern after TPA exposure in spite of a strongly suppressed GJIC [8, 11]. Okadaic acid and calyculin A do not affect the normalization of GJIC in SHE cells after TPA preexposure [8]. We
therefore investigated whether cyclosporin A or FK506 could affect the normalization of GJIC after preexposure of SHE cells to TPA (150 nM). The normalization in SHE cells occurred faster than in V79 cells. There was only a short lag phase after the removal of TPA, since GJIC had started to increase already at 30 min and was back at control level at 2 h (not shown). In the continued presence of TPA (150 nM), GJIC recovered much slower and was around 50% of control after 8 h of exposure (not shown). FK506 delayed the normalization of GJIC during the first part of the period after removal of TPA (P , 0.05; not shown). Also cyclosporin A appeared to slightly delay the normalization (not shown), but the effect was not statistically significant at any point of time. If the PP2B activity was of importance for the normalization of GJIC, we should expect rapamycin not to affect the recovery of GJIC after TPA exposure. Rapamycin (up to 1000 nM) alone did not affect GJIC (Fig. 3A), and it did not delay the recovery after TPA exposure in V79 cells (Fig. 3A). In contrast, 1000 nM slightly enhanced the recovery rate during the first 3 h after removal of TPA (Fig. 3A). Since rapamycin and FK506 compete for binding to FKBPs, rapamycin should counteract the effect of FK506 [28, 29]. At concentrations of 300 to 1000 nM, rapamycin counteracted the effect of 300 nM FK506 during 3 to 4 h of coexposures after removal of TPA. This is exemplified by the results for 3.5 h shown in Fig. 3B. Cx43 Band Pattern The Cx43 bands were named according to previously used nomenclature [11, 22, 30]. V79 cells that had been growing for 24 – 48 h showed two strong Cx43 bands (Fig. 4A). The lower strong band corresponded to the nonphosphorylated (NP) form of Cx43, as it aligned with Cx43 dephosphorylated by alkaline phosphatase (not shown). The slower migrating strong band was called P1. There were also two weaker bands, the P9 form migrating immediately above NP, and P2 migrating above P1. V79 cells modify their Cx43 band pattern in response to addition of fresh growth medium as described elsewhere [31], but not to addition of conditioned medium (Fig. 4A). The results shown in Figs. 4 and 5 were obtained with conditioned medium, but we came to the same conclusions using fresh growth medium. TPA induced relatively large changes in the V79 – Cx43 band pattern (Fig. 4B, 0 h recovery time) as described before [8, 11]. The P2 band became especially intense and was the strongest band under these conditions. In addition, a slow-migrating HP form was found above P2. After removal of TPA, the strong changes in the band pattern mainly reversed within 2–3 h (Fig. 4B). The HP band disappeared, the P2 band
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V79 cells were then exposed to okadaic acid, calyculin A, cyclosporin A, FK506, or rapamycin for 1 to 4 h before sampling. None of the compounds had any effect on the band pattern of Cx43 as exemplified by calyculin A (Fig. 5A) and FK506 (Fig. 5B). Okadaic acid (100 nM) and calyculin A (3 nM) did not affect the normalization of Cx43 band pattern after removal of TPA (not shown), suggesting that PP1 and PP2A are not among the major phosphatases involved in dephosphorylation of Cx43 in V79 cells under these conditions. Conversely, both cyclosporin A and FK506 delayed the normalization of the TPA-induced changes in Cx43 band pattern. This was most evident for FK506 (Fig. 4C). The compounds extended somewhat the presence of the HP band and the strong P2 band and delayed the appearance of the NP band and P1 band. The presence of the doublet P1/P19 was extended to at least 3.5 to 4 h, and P9 remained relatively strong during the whole 6-h recovery period. Rapamycin (300 and 1000 nM) delayed the normalization of band pattern (Fig. 4D) even more than did FK506.
FIG. 3. Effects of rapamycin on GJIC in V79 cells. (A) Influence of rapamycin during the recovery period after preexposure of V79 cells to TPA. Cells were pretreated with growth medium for 30 min, rinsed, and exposed to (‚) 300 or (ƒ) 1000 nM rapamycin. Alternatively, cells were preexposed to 5 nM TPA for 30 min, rinsed, and added (h, dashed curve) growth medium only or (Œ) 300 nM rapamycin and () 1000 nM rapamycin for the indicated periods before measuring GJIC (n 5 38 –71). Rapamycin (1000 nM) slightly enhanced the recovery rate at 2 to 3 h after removal of TPA (P , 0.05). (B) Rapamycin counteracts the effect of FK506. V79 cells were exposed to 5 nM TPA for 30 min. The medium was then removed, and the cells were allowed to recover for 3.5 h in medium containing (bar 1) only growth medium (n 5 49); (bar 2) 300 nM FK506 (n 5 57); (bar 3) 300 nM FK506 and 300 nM rapamycin (n 5 48); or (bar 4) 300 nM rapamycin (n 5 13). The data are shown as means 6 SEM. Statistical significances: P , 0.001 for bar 1 vs. unexposed control, bar 1 vs. bar 2, and bar 2 vs. bar 3.
became less intense, and the NP and P1 bands gradually regained their initial intensities. A double band, P1/P19, was seen in the period from 1 to 2 h. It should be noted that the extent of TPA-induced V79 –Cx43 band pattern changes and their reversal rates varied to some degree with cell confluence (not shown). Cycloheximide did not affect the Cx43 band pattern during 4-h exposures (not shown). It did also not change the rate of normalization of Cx43 band pattern after removal of TPA (not shown). This indicated that dephosphorylation of Cx43 must be a major mechanism for reversal of the TPA-induced changes in V79 – Cx43 band pattern.
FIG. 4. FK506 and rapamycin affected the normalization rate of Cx43 band pattern in V79 cells after removal of TPA. V79 cells were subjected to (A) changes of conditioned growth medium only, or they were preexposed to 5 nM TPA for 30 min before rinsing and then added (B) conditioned growth medium only, (C) 300 nM FK506, or (D) 300 nM rapamycin. The normalization was followed up to 6 h after removal of TPA. The positions of the nonphosphorylated (NP) form and the phosphorylated forms (P9, P1, P19, P2, HP) of V79 – Cx43 are shown to the right. P, prestained standards with molecular mass (in kDa) given to the left. Pre, sample without any change of medium.
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FIG. 5. Lack of effect of phosphatase inhibitors on Cx43 band pattern in V79 cells. (A) The cells were exposed to 3 nM calyculin A for the indicated periods. Note that the cells started to round up already after 30 min. The cells were therefore rinsed very carefully before sampling to avoid losses of cells. (B) The cells were exposed to 300 nM FK506 for the indicated periods.
Immunorecognition of Cx43-Containing Gap Junctions in V79 Cells Immunofluorescence experiments were performed to examine the effects of the compounds on immunorecognition of gap junction plaques in cells. V79 cells had relatively few gap junctions at cell– cell borders (Fig. 6A) compared with many other cell types [see also Ref. 26], even using the present anti-Cx43 antiserum [32, 33]. In addition to typical gap junction plaques, there were some intracellular Cx43-positive spots and a perinuclear distribution of Cx43. The perinuclear population of Cx43 and its importance in Cx43 band pattern alterations after stimulation of cells were recently described [31].
As has been observed previously [11, 32], TPA decreased the recognition of gap junction plaques in immunofluorescence experiments (Fig. 6C). Interestingly, this effect became even more prominent during the first hour after removal of TPA (Fig. 6D). At 2–2.5 h after TPA removal, the recognition of Cx43 started to reappear, and it increased up to control level during the next 1.5 h (Figs. 6C– 6E, 7A, and 7B). The procedure of rinsing and change of growth medium (fresh or conditioned) did not cause any change in immunorecognition of gap junction plaques under the present conditions (not shown). During prolonged exposure to TPA, the gap junction recognition continuously decreased during the first 1.5–2 h similar to the curves shown in Figs. 7A and 7B, but then stayed low for several hours. Only at 6 h, some reappearance of gap junctions occurred. Cycloheximide alone did not affect the amount of gap junction plaques (Fig. 7B), and it did not change the kinetics of recovery after removal of TPA (Fig. 7B). Thus, the gap junction reappearance also seemed independent of protein synthesis. The phosphatase inhibitors or rapamycin per se did not affect the amount of gap junction plaques (Figs. 7A and 7B; and data not shown) or localization of Cx43 in cells, as exemplified by FK506 (Fig. 6B). Calyculin A and okadaic acid did not change the return kinetics of gap junction plaques (not shown). Cyclosporin A appeared to slightly delay the return of gap junctions, but the effect was more pronounced with FK506 (compare Figs. 6E and 6F; see also Fig. 7A). Visually, the return
FIG. 6. Immunorecognition of Cx43 in V79 cells. V79 cells were preexposed to growth medium for 30 min, rinsed, and further exposed to (A) growth medium or (B) 300 nM FK506 for 4 h. (C) Cells were exposed to 5 nM TPA for 30 min. (D and E) Cells were preexposed to 5 nM TPA for 30 min, before rinsing and recovery in the presence of only growth medium for (D) 1 h and (E) 3.5 h. Alternatively, the cells were exposed to (F) 300 nM FK506 during the recovery period of 3.5 h. These micrographs are a part of the basis for the curves shown in Fig. 7. Bar in A, 25 mm.
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seemed to be delayed at least by 30 min to 1 h. This was confirmed by quantitation of the gap junction plaques (Fig. 7A). Rapamycin had a more potent effect than FK506 and strongly inhibited the recovery of gap junction plaques both at 300 nM (Fig. 7B) and 1000 nM (not shown). To investigate if the size distribution of the gap junctions was altered during the treatments, the gap junctions were divided into three size groups as described under Materials and Methods. The size distributions of the plaques, whose densities are depicted in Fig. 7B, are shown in Fig. 7C. A statistical analysis was performed on these size distributions and for a similar set of data from the experiments shown in Fig. 7A. Only the most important conclusions will be mentioned here. The analysis indicated time-dependent trends in size distributions after exposure to TPA. In the period from 1 to 2 h, there was a significant reduction in percentage of large plaques in the TPA preexposed group, even when corrected for the nonsignificant reduction of large plaques in the control samples. Neither cyclosporin A, FK506 (not shown), cycloheximide, nor rapamycin alone (Fig. 7C) caused significant changes in gap junction size distribution. However, in the period from 2.5 to 4 h after removal of TPA, rapamycin significantly inhibited the normalization of the size distribution (Fig. 7C). A similar, but not significant, tendency was also seen for the exposure to TPA followed by FK506. For the sequential exposures to TPA followed by growth medium, cyclosporin A, or cycloheximide, there were significant time-dependent recoveries toward control values, with faster recoveries of small and medium sized gap junctions.
FIG. 7. Gap junction plaque recovery in V79 cells. (A) Influence by cyclosporin A and FK506. V79 cells were preexposed to 5 nM TPA for 30 min, rinsed, and added (h, dashed curve) growth medium only, (■) 300 nM FK506, or (F) 300 nM cyclosporin A. Alternatively, they were added new growth medium for 30 min and then changed to growth medium containing (h) 300 nM FK506 or (E) 300 nM cyclosporin A. Unexposed control cultures were only added growth medium. Cell cultures were fixed at the indicated times after rinsing and processed for immunofluorescence, and gap junction plaques were quantified as described under Materials and Methods. At any time point, the unexposed control cultures only added growth medium were defined as the 100% level of number of gap junctions per unit length of membrane. The controls varied between 0.77 and 0.87 identifiable gap junctions per 10 mm of cell– cell border membrane. For clarity, the deviations are not shown, but for most points
SEM was ,10% of the mean. Statistical significances (arrow indicates the sequential exposures): TPA 3 DMEM vs. DMEM 3 DMEM, P , 0.001 at 0, 1, 2, 2.5, and 3 h; TPA 3 cyclosporin A vs. DMEM 3 cyclosporin A, P , 0.001 at 2.5, 3, and 3.5 h; TPA 3 cyclosporin A vs. TPA 3 DMEM, P , 0.05 at 3.5 h; TPA 3 FK506 vs. DMEM 3 FK506, P , 0.001 at 2.5, 3, 3.5, and 4 h; TPA 3 FK506 vs. TPA 3 DMEM, P , 0.01 at 3.5 and 4 h. (B) Influence of rapamycin, but not cycloheximide, on gap junction recovery. V79 cells were preexposed to 5 nM TPA for 30 min, rinsed, and added (h, dashed curve) growth medium only, (Œ) 10 mM cycloheximide, or () 300 nM rapamycin. Alternatively, they were added new growth medium for 30 min and then changed to growth medium containing (‚) 10 mM cycloheximide or (ƒ) 300 nM rapamycin. Unexposed control cultures were only added growth medium. Statistical significances: TPA 3 DMEM vs. DMEM 3 DMEM, P , 0.001 at 0, 1, 2, and 2.5 h; TPA 3 cycloheximide vs. DMEM 3 cycloheximide, P , 0.001 at 2.5 h; TPA 3 rapamycin vs. DMEM 3 rapamycin, P , 0.001 at 2.5, 3, 3.5, and 4 h; TPA 3 rapamycin vs. TPA 3 DMEM, P , 0.001 at 2.5, 3, 3.5, and 4 h. (C) Gap junction size distribution (mean 6 SEM) for the density data shown in B. Gap junction size was classified as described under Materials and Methods. The percentages of small gap junctions are shown by filled bars, medium sized gap junctions by hatched bars, and large gap junctions by open bars. For simplicity, the values for 1–2, and 2.5– 4 h were grouped. CHX, 10 mM cycloheximide; R, 300 nM rapamycin.
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TABLE 1 Effects in V79 Cells of the Studied Compounds on GJIC, Cx43 Phosphorylation, and Immunorecognition of Cx43Containing Gap Junction Plaques in the Recovery Phase after a Short Preexposure to TPA Delay in recovery after 30 min of exposure to 5 nM TPA
Compound
GJIC
Cx43 phosphorylation
Cx43 immunorecognition
Cycloheximide Okadaic acid Calyculin A Cyclosporin A FK506 Rapamycin
0a 0 0 1a 1 0
0 0 0 1 11 111
0 0 0 1 11 111
a
No effect is indicated by 0, and an increasing effect on delay is indicated by increasing numbers of pluses.
The mechanisms of gap junction disappearance are unclear, but may include both fragmentation of gap junctions and epitope masking. In the recovery phase from 2.5 to 4 h, the channels probably first assembled into smaller gap junction plaques. The effects of the studied compounds on the recognition of gap junction plaques in the recovery period after TPA exposure are summarized in Table 1, together with the results for GJIC and Western blots of Cx43. Presence and Activities of Some PPs The presence of PP1, PP2A, and PP2B in V79 cells was investigated by Western blots. Anti-PP1a (two antibodies), anti-PP1b, anti-PP1g, and anti-PP2A (two antibodies) gave weak bands in the expected area around 36 –37 kDa (not shown), but none of the antibodies were monospecific. Anti-PP2B recognized a prominent band around 60 kDa, the expected position of the catalytic subunit (Fig. 8). For measurement of PP1 and PP2A activities in the cells, 32P-labeled phosphorylase a was used as substrate. The PP1 activity in both V79 and SHE cells averaged 80 – 85% of the total phosphorylase a phosphatase activity when low amounts of protein (0.2– 0.4 mg/sample) were used (Table 2). Using higher amounts of protein, the PP1 activity decreased relative to the PP2A activity (not shown). Still calyculin A was able to totally inhibit all phosphorylase a phosphatase activity at 10 nM, while okadaic acid inhibited 85% of the activity at 100 nM when included in the assay. The major phospho-RII phosphatase activities were due to PP1/PP2A and PP2B. The PP1/PP2A activity was determined by the sensitivity to calyculin A and okadaic acid and was approximately twice the activity of PP2B (Table 2), determined by the sensitivity to the combination of FK506 and FKBP.
The major phospho-casein phosphatase activity appeared to be PP2A (Table 2 and data not shown), in line with previous results [24]. However, the specific activity of PP1/PP2A was only around 2% relative to that measured using phosphorylase a as substrate (Table 2). PP2C activity was 15–30% relative to the PP1/PP2A activity with casein as substrate (Table 2). Thus, also PP2B and PP2C have considerable activities in these cell systems. However, the importance of PP2C in intact cells is difficult to assess as no specific inhibitors are known. The regulatory mechanisms for PP2C are unclear, and the activation of PP2C by a high Mg 21 concentration (20 mM) in the enzymatic assay is probably not physiologically relevant. Dephosphorylation of Immunoprecipitated Cx43 Cx43 was immunoprecipitated from V79 cells. In these experiments, V79 control cells were not subjected to any change of medium. Therefore they showed a low amount of the P2 band. In addition, as has been observed by others [30], the P2 band can often be difficult to fully solubilize for immunoprecipitation. Alkaline phosphatase efficiently removed phosphate groups from Cx43 and left only the NP form detectable (not shown; [8, 31]). We have investigated here the Cx43 dephosphorylation efficiencies by different phosphatases. Cx43 immunoprecipitates were treated with PP1 (g isoform, catalytic subunit; Fig. 9A), PP2A (catalytic subunit; Fig. 9B), PP2B (holoenzyme; Fig. 9C), or PP2C (a isoform; Fig. 9D). PP2A was clearly more active than the other phosphatases in dephosphorylating Cx43 from untreated V79 cells (Fig. 9). Three or four concentrations of PP2A were therefore used in all experiments for comparison with the other phosphatases since the amount of immunoprecipitated Cx43 may vary somewhat between the experiments. In most experiments, a band in the P9 position could be seen in addition to the NP form, but in a few experiments only the NP form of Cx43 was detectable after PP2A treatment. The P9 band was generally more prominent in immunoprecipitates treated with PP1 (Fig. 9A), PP2B (Fig. 9C), and PP2C (Fig. 9D). In addition, weak bands in the upper part of the Cx43 area could often be seen in PP1-, PP2B-, and especially PP2C-treated immunoprecipitates. Taking into account the concentrations of the phosphatases, PP2A was about 100-fold more active on
FIG. 8. Detection of catalytic subunit of PP2B in V79 cells by Western blot.
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PHOSPHATASES AND CONNEXIN43
TABLE 2 PP1, PP2A, PP2B, and PP2C Activities in V79 and SHE Cells Phosphatase (substrate used)
V79 cells [nmol P i released/min/mg protein (n)]
SHE cells [nmol P i released/min/mg protein (n)]
PP1 (phosphorylase a) a PP2A (phosphorylase a) a PP1/PP2 (RII peptide) b PP2B (RII peptide) b PP1/PP2A (casein) c PP2C (casein) c
7.29 6 0.57 (5) 1.56 6 0.17 (5) 4.95 6 0.73 (5) 2.04 6 0.76 (5) 0.199 6 0.036 (3) 0.034 6 0.010 (6)
6.79 6 1.08 (3) 1.46 6 0.19 (3) 4.97 6 0.50 (5) 2.40 6 0.45 (6) 0.142 6 0.016 (2) 0.041 6 0.002 (3)
a The activities were determined by using 32P-labeled phosphorylase a as substrate in strongly diluted homogenates of cells (0.2– 0.4 mg cell protein/sample). The PP1 part of the activity was determined by using inhibitor-2, and the remaining phosphatase activity is regarded as PP2A. The plateau of inhibition was reached at 3 nM inhibitor-2 under this condition. The data are shown as means 6 SD. b PP2B was regarded as the phospho-RII phosphatase activity that could be inhibited by the combination of 50 mM FK506 and 10 mM FKBP. PP1/PP2A activity was regarded as the phospho-RII phosphatase activity that could be inhibited by 10 –100 nM calyculin A or okadaic acid, using 2.5–5 mg cell protein/sample. The data are shown as means 6 SD. c PP2C activity was regarded as the Mg 21-activated phospho-casein phosphatase activity. PP1/PP2A activity was regarded as the phospho-casein phosphatase activity that could be inhibited by 10 –100 nM calyculin A or okadaic acid, using 1.5–5 mg cell protein/sample. The data are shown as means 6 SD.
phospho-Cx43 than PP1, PP2B, and PP2C (Table 3). Control experiments showed that the dephosphorylation of Cx43 by PP2A was inhibited by okadaic acid and calyculin A (Fig. 9E). For PP2B, FKBP was necessary for the inhibition by FK506 (Fig. 9E). The present antiserum is directed against residues 364 –382 in the rat Cx43 sequence. This area contains at least two potential phosphorylation sites [15]. The relatively phosphatase-resistant P9 band could therefore be due to phosphorylation sites protected by the antiserum. We believe that this possibility is unlikely based on the following results: (i) Some Cx43 was released from the beads during the PP1, PP2A, and PP2B treatment (not checked for PP2C), probably due to the content of DTT or b-mercaptoethanol in the buffers causing denaturing of the immunoglobulins. Also the released Cx43 contained the P9 band (not shown). (ii) In separate experiments, the immunoprecipitate was boiled (in PP2B buffer), and the supernatant was treated with PP2B. The P9–Cx43 band was still present (not shown). Cx43 immunoprecipitates from TPA-exposed V79 cells were also treated with phosphatases. PP2A dephosphorylated Cx43 from TPA-treated cells with approximately the same efficiency as Cx43 from control cells (Fig. 9B). PP1 seemed to be less efficient in dephosphorylating the P2–Cx43 band from TPA-treated cells than the same band from control cells (Fig. 9A), but if the amount of Cx43 was lowered, dephosphorylation was achieved (not shown). This supports our recent observation that TPA exposure of V79 cells generates a P2–Cx43 band that is different from the P2– Cx43 band in unstimulated cells [31]. PP2B (Fig. 9C) and especially PP2C (Fig. 9D) were also clearly less active on Cx43 immunoprecipitated from TPA-treated cells. TPA may induce tyrosine phosphorylation of
some proteins [34 –36]. We therefore considered the possibility that the resistance was due to tyrosine phosphorylation of Cx43, although PP2B has a quite high phosphotyrosine phosphatase activity [37] and also PP2A may act on phosphotyrosine to a lesser extent [38, 39]. However, Cx43 from TPA-exposed V79 cells did not contain phosphotyrosine as detected with the anti-phosphotyrosine antibody 4G10 (not shown). This is consistent with previous results [20, 31]. Altogether, these results indicate both quantitative and qualitative differences in activities between phosphatases in dephosphorylation of Cx43. DISCUSSION
As detected by Western blots, the phosphorylation status of Cx43 varies between different tissues [40], in different cultured cell types [8, 31], and as a response to many stimuli. This may suggest that different kinases and/or phosphatases act on Cx43 in different cells and that several combinations of phosphorylation sites in Cx43 are possible. In the present work, the PKC activator TPA was used as a model compound to induce decreased GJIC, altered phosphorylation of Cx43, and diminished immunorecognition of Cx43-containing gap junction plaques in V79 cells. Protein synthesis was not necessary to obtain reversal for any of the three cellular parameters, suggesting the involvement of phosphatases. This involvement was approached from two sides: (i) we investigated the influence of several phosphatase inhibitors on the three cellular parameters after exposure to TPA, and (ii) we assessed the phosphatase activities in the cells and studied the efficiency of the major types of phosphatases to dephosphorylate immunoprecipitated Cx43 from control and TPA-treated V79 cells.
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TABLE 3 Approximate Cx43 Dephosphorylating Activities of Phosphatases Relative to PP2A Relative phospho-Cx43 phosphatase activities based on
PP1 PP2A PP2B PP2C
Molarities a
Specific enzyme activities (protein substrates) b
Specific enzyme activities (pNPP) c
0.3 100 d 1 ,1
0.05 100 d — ,10
1 100 d 10 —
a
Calculated from the phosphatase concentrations shown in Fig. 9. Calculated from specific protein phosphatase activities given by the supplier. The protein substrates were: PP1 and PP2A, phosphorylase a; PP2C, casein. c Calculated from the specific para-nitrophenyl phosphate (pNPP) phosphatase activities given by the supplier. d The phospho-Cx43 phosphatase activity of PP2A is arbitrarily defined as 100. b
FIG. 9. Dephosphorylation of immunoprecipitated Cx43 from V79 cells. Cx43 from control cells or cells exposed to 5 nM TPA for 30 min was immunoprecipitated and treated with phosphatases for 3 h at 37°C as described under Materials and Methods. The band pattern of Cx43 was studied by Western blotting. The apparent molecular mass (in kDa) of the prestained standard (lane marked with P) is shown to the left. The positions of the NP, P9, P1, and P2 forms of Cx43 are also marked to the left. The pronounced band at around 55 kDa is the heavy chain of the immunoprecipitated immunoglobulins. (A) PP1. The immunoprecipitates were treated with PP1 buffer only (lanes marked 0) or 0.05, 0.16, 0.54, and 1.62 mM PP1. For comparison, the Cx43 band patterns from total homogenates (Hom) of control cells, TPA-treated (5 nM, 30 min) cells, and nontreated immunoprecipitates (IP) are also shown. (B) PP2A. The immunoprecipitates were treated with PP2A buffer only (lanes marked 0) or 1.9, 5.6, and 18.5 nM PP2A. The blot shown was obtained from the same experiment as the blot shown in A. (C) PP2B. Immunoprecipitates were treated with PP2B buffer only (lanes marked 0) or 0.17, 0.57, and 1.7 mM PP2B. (D) PP2C. Immunoprecipitates were treated with PP2C buffer only (lanes marked 0) or 70, 299, and 698 nM PP2C. The Cx43 bands of the control immunoprecipitate treated with 70 nM
The phosphorylation sites in Cx43 from intact cells are not known, but peptide-based kinase assays suggest two main clusters of potential phosphorylation sites. (i) Several serines in PXSP sequences between positions 253 and 283 may be phosphorylation sites for mitogen-activated protein kinase [41] or p34 cdc2 [42]. (ii) Close to the C-terminal tail, serines 368 and 372 were found to be phosphorylated by PKC [15]. Since PKC also may mediate the activation of the mitogenactivated protein kinase pathway [see Refs. 43, 44], there may be changes in the phosphorylation status in both clusters in addition to other unknown phosphorylation sites in response to TPA. PP1 [45, 46] and PP2C [47] perceive a proline in position 11 as a strong negative determinant. PP1 and PP2C are therefore not likely to dephosphorylate the potential phosphoserines in the PXSP sequences (positions 255, 279, and 282). Some proline-containing sequences can be good substrates for PP2A and PP2B [46 – 49]. Thus, we would expect PP2A and PP2B to be
PP2C are weak, but on the original blot it can be clearly seen that the band pattern is similar to that of the corresponding nontreated immunoprecipitate (IP) or buffer-treated immunoprecipitate (lanes marked 0). (E) Inhibition of PP2A- (left) or PP2B- (right) mediated Cx43 dephosphorylation. Immunoprecipitates were treated with the PP2A buffer (0), 55.5 nM PP2A, or 55.5 nM PP2A with 555 nM okadaic acid (OA) or 555 nM calyculin A (Cal). Okadaic acid or calyculin A was mixed with the enzyme before addition to the immunoprecipitate. Immunoprecipitates were also treated with PP2B buffer (0), 1.7 mM PP2B, or 1.7 mM PP2B with FKBP (2 mM) and FK506 (20 mM) (FK/FKBP). As controls, it is shown that FKBP and FK506 (FK) were not able to inhibit PP2B when they were added separately. Also shown are Western blots of total homogenates (Hom) and nontreated immunoprecipitates (IP) from the corresponding experiments.
PHOSPHATASES AND CONNEXIN43
the more active Cx43 phosphatases, while PP1 and PP2C might be less active. The studies on dephosphorylation of immunoprecipitated Cx43 seemed to partially support the above expectation. All the four phosphatases studied were active in dephosphorylating immunoprecipitated Cx43 from control cells, but PP2A was about 100-fold more efficient than were the other phosphatases (Table 3). In comparison, PP2A is approximately 30-fold more active than PP1 in dephosphorylating pp54 microtubule-associated protein 2 kinase [50, 51]. Interestingly, only PP2A, and to some degree PP1, were able to dephosphorylate Cx43 from TPA-treated cells, while PP2B and PP2C were nearly inactive. It is unlikely that the lack of PP2B and PP2C action is caused by protection of phosphorylation sites by the antibodies. The most reasonable alternative is therefore that the amino acid sequences around certain TPA-induced phosphorylations render these sites less attractive for PP2B and PP2C. The high efficiency of PP2A in dephosphorylation of immunoprecipitated Cx43 was somewhat surprising since okadaic acid and calyculin A did not affect GJIC or Cx43 band pattern, whether the compounds were applied alone or in sequential exposures after TPA (Table 1). These compounds are well known as inhibitors of PP1 (IC 50 10 –15 and 2 nM for okadaic acid and calyculin A, respectively) and PP2A (IC 50 0.1 and 1 nM for okadaic acid and calyculin A, respectively). In addition, okadaic acid inhibits several other more recently identified phosphatases, e.g., PP3 (IC 50 5 nM) [52], PP4 (IC 50 , 0.2 nM), PP5 (IC 50 , 2 nM), and PP6 (IC 50 , 2 nM) [53]. The lack of effects of the PP1/PP2A inhibitors on GJIC in V79 cells is fully in line with previous results obtained in SHE cells [8, 13]. Thus, it is not likely that these phosphatases are involved to any major extent in the regulation of phosphorylation status of Cx43 or GJIC under basal conditions or during the normalization phase after stimulation with TPA in the present cell culture systems. This is in contrast to the results of Lau et al. [9], who found 1 nM okadaic acid to delay normalizations of Cx43 phosphorylation and GJIC after exposure of T51B rat liver epithelial cells to EGF. It is not known if the differences are due to the stimulating agent (TPA vs. EGF) or the experimental systems used. However, okadaic acid or calyculin A minimally affect the normalization of serum-induced changes in V79 –Cx43 band pattern (V. Cruciani and S.-O. Mikalsen, unpublished results). The lack of effect by calyculin A and okadaic acid was not due to lack of PP1 and PP2A in the cells, since they could be detected by both enzyme measurements and Western blotting. It was previously shown that the catalytic subunit of PP2A had altered dephosphorylation activities toward several substrates compared to the holoenzyme [48, 54]. In addition, proteins may interact with phosphatases in more complex manners
459
than peptides [46], as different subunits and factors present in cells may change specificities and activities of phosphatases relative to those seen in cell-free systems. This is further confirmed by the variation in PP1/PP2A activities in our phosphatase measurements (Table 2). However, in independent experiments we verified that a PP2A preparation containing both catalytic and regulatory subunits (Upstate Biotechnology) dephosphorylated Cx43 from control cells at very similar concentrations as did the Boehringer PP2A catalytic subunit. Thus, other explanations should be offered for the differences between cells and the cell-free system. The accessibility of the phosphorylation sites might differ. This could be due to altered three-dimensional structure of Cx43 (denatured in the immunoprecipitates), targeting of phosphatases to specific locations in the intact cells by anchoring proteins [reviewed in 55], or that accessory proteins may protect phosphorylated Cx43 residues against dephosphorylation in intact cells. The two clusters of potential phosphorylation sites in Cx43 correspond to potential target sequences for accessory proteins and coupling factors like 14-3-3 proteins and WW-, SH2-, and SH3-domain proteins [11, 23]. At variance with the lack of effect of okadaic acid and calyculin A, two inhibitors of PP2B, cyclosporin A and FK506, delayed normalization both of GJIC, Cx43 band pattern, and immunorecognition of gap junction plaques (Table 1). The cells contained a considerable amount of PP2B as detected both by Western blot and by enzyme measurements. We have also used the asserted PP2B inhibitor cypermethrin (IC 50 40 pM) [56]. It was previously shown that cypermethrin decreased dye transfer in BALBc3T3 cells at 20 mM or higher concentrations [57], but it did not inhibit metabolic cooperation in V79 cells [58]. In our hands, cypermethrin did not affect GJIC, Cx43 phosphorylation or Cx43 immunorecognition, whether it was used alone or in sequential combination with TPA (results not shown). It also did not affect the dephosphorylation of immunoprecipitated Cx43 by PP2B. Our results therefore suggest that cypermethrin is not an inhibitor of PP2B or is of very low potency. Other groups recently came to the same conclusion [59, 60]. Inhibition of PP2B by cyclosporin A or FK506 is dependent on the compound-induced complexing of immunophilins with PP2B [17]. Relatively little is known about the cellular functions of immunophilins. All of them appear to possess PPIase activity that is inhibited by the compounds [18, 61– 63]. FKBPs and cyclophilins may participate in protein folding [62, 64] and possibly in trafficking and secretion of proteins [65, 66]. In addition, some immunophilins are found associated with membrane proteins, including membrane channels [67, 68]. We therefore considered the possibility that the effects of FK506 and cyclosporin A might
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be due to inhibition of functions of the FKBPs and cyclophilins and not directly associated with PP2B inhibition. Rapamycin competes with FK506 for binding to FKBPs [28, 29], and it inhibits the PPIase activity of the FKBPs, but it does not inhibit PP2B [17, 19]. Thus, rapamycin makes it possible to distinguish between these two enzyme activities. Rapamycin did not delay the recovery of GJIC after preexposure to TPA, but it counteracted the delaying effect of FK506 on GJIC. Altogether, this indicates that PP2B is involved (directly or indirectly) in the recovery of GJIC after TPA exposure in the present cell systems. This conclusion does not exclude the possibility that other phosphatases also are involved in this process. In intriguing contrast to the lack of effect on GJIC, rapamycin clearly delayed the normalizations of Cx43 band pattern and immunorecognition similarly to cyclosporin A and FK506 (Table 1). The consistency in effect by the three compounds in this respect makes it less likely that rapamycin-specific influences on signal transduction [69] are involved. These results may suggest that PP2B does not participate in the dephosphorylation of Cx43 after TPA exposure, as also supported by the low efficiency of PP2B to dephosphorylate Cx43 from TPA-exposed cells (Fig. 9C). Alternatively, the dephosphorylation of Cx43 by PP2B may depend on the PPIase activity of the immunophilins. Some immunophilins may show specificity for phosphorylated residues adjacent to prolines, and it has been speculated that PPIase activity may be necessary to make these phosphoamino acids more sensitive to phosphatases [70]. To investigate if PPIase activity could promote dephosphorylation of Cx43 by the phosphatases, FKBP was added together with PP1, PP2A, or PP2B in the dephosphorylation assay. No enhancement of dephosphorylation was obtained (results not shown). Since the used FKBP is included in a family consisting of at least seven members (also the cyclophilin family consists of multiple members) [71], this does not exclude the possibility that other combinations of phosphatases and immunophilins could promote dephosphorylation of Cx43. There are a number of studies on the potential interactions between PKC and PP2B. The enzymes may synergize [72] or counteract each other [73, 74], depending on the context. In some systems, both enzymes bind to the same anchoring protein, AKAP79, which also binds protein kinase A [75]. Furthermore, cyclosporin A and FK506 have previously been shown to inhibit TPA-induced effects in vivo, including mouse skin tumor promotion [76, 77]. Cyclosporin H, a compound that does not bind to immunophilins, is also a potent inhibitor of TPA-induced effects in vivo [78], suggesting that PP2B may not be involved in cyclosporin-induced inhibition of TPA effects in vivo. In addition, at least some of the TPA-induced effects in
mouse skin are not dependent on T lymphocytes [79]. Cyclosporin A may also inhibit proteolytic cleavage of activated PKC [80]. Thus, the biological interactions between these enzymes and/or compounds are not straightforward. The present results give support to the view that there is not a direct correlation between effects on GJIC and the changes in phosphorylation status of Cx43, even under conditions that activate kinases or inhibit phosphatases [8, 11, 21, 23, 31]. This is especially evident for the results obtained with rapamycin. A part of the dissociation between the two parameters may be explained by phosphorylation of an intracellular subpopulation of Cx43 [31]. On the other hand, the potential correlation between the Cx43 band pattern and gap junction plaque recoveries after TPA exposure may deserve a closer investigation. We have previously suggested that TPA induces the binding of accessory proteins to Cx43, thus contributing to epitope masking [11], although it may be difficult to distinguish between epitope masking and dispersal of Cx43 in the cell membrane by the present methods. It is possible that such accessory proteins participate in the regulation of GJIC and that these proteins are targets for PP2B. Still it is likely that phosphorylation and changes in phosphorylation status of Cx43 by experimental and physiological agents (e.g., lysophosphatidic acid, growth factors, several hormones) or by physiological conditions [81] are relevant for some aspects of Cx43 function and regulation. The enhancement of the later stages of tumor development by cyclosporin A is well known, while very little is known for the other immunosuppressive agents used here. Some of the tumor-enhancing mechanisms of cyclosporin A appear independent of the direct immunosuppressive properties [82]. Effects on gap junctions may add to the nonimmunosuppressive biological effects of these compounds. We thank Dr. T. Sanner for valuable discussions and Dr. E. Rivedal for making the anti-Cx43 antiserum available to us. This work was supported by Jahre’s Fund (to T. Sanner and S.-O.M.), The Family Blix’ Fund (to V.C. and S.-O.M.), and funds from The Medical Faculty, University of Oslo (to V.C. and S.-O.M.), and the Norwegian Cancer Society (to T. Sanner, E. Rivedal, and S.-O.M.).
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